The IL-6 stimulated terminal differentiation of murine myeloid leukemia M1 cells, into mature macrophages, represents a unique model to dissect molecular and cellular events regulating myeloid differentiation. Expression of inducible nitric oxide synthase (iNOS) constitutes one of the classical hallmarks of a functionally mature macrophage. This enzyme catalyzes the synthesis of nitric oxide (NO), which mediates the antitumour and antibacterial responses of macrophages. In order to address the nature of trans-acting factors participating in the IL-6 stimulated transcriptional activation of the iNOS gene, we focussed on Region I in the proximal 5' region of the iNOS promoter. This region contains several essential elements mediating the transcriptional activation of the iNOS gene in response to LPS. A candidate, non consensus, octamer sequence has been identified 31bp upstream of the TATA box in the iNOS gene. IL-6 stimulation led to the induction of DNA binding proteins which recognise an Oct-1 binding site consensus sequence. The time-dependent appearance of these DNA-protein complexes correlated well with the kinetics of iNOS mRNA expression. Methylation interference analysis reveals that in IL-6 treated M1 cells, the octamer sequence was bound by proteins which protected a single G residue. We also found that this octamer sequence is absolutely required for iNOS promoter activity in IL-6 stimulated M1 cells stably transfected with a luciferase reporter construct. Supershift assays and antibody inhibition experiments verified that IL-6 stimulation led to differential regulation of Oct-1, Oct-2, as well as several other octamer binding proteins. iNOS promoter fragments harboring mutated octamer motifs, in the context of an otherwise wild-type sequence, were shown to be incapable of binding to IL-6 induced transacting factors.